N-acylethanolamines from fatty acids are an important class of alkanolamides that function as nonionic surfactants and have a wide range of applications in the lubricants, surfactants and detergents, cosmetics, and other industries (Liu et al., J. Agric. Food. Chem. 49:5761-64 (2001); Sanders, J. Am. Oil Chem. Soc. 35:548-51 (1958); Feairheller et al., J. Am. Oil Chem. Soc. 71:863-66 (1994)). Certain N-acylethanolamines are lipid mediators in animals and plants (Kilaru et al., Chem. Biodivers. 4:1933-55 (2007); Re et al., Vet. J. 173:21-30 (2007); Terrazzino et al., FASEB J. 18:1580 (2004)). It has been reported that N-acylethanolamines of different chain length and structure exhibited a variety of biological activities. N-palmitoylethanolamine had anti-inflammatory activity, and attenuates pain sensation (Re et al., Vet. J. 173:21-30 (2007); Calignano et al., Nature 394:277-81 (1998)). It was also shown to reduce allergic reaction, inhibit mast cell degranulation (Aloe et al., Inflamm. Res. 39:145-47 (1993)) and exert neuroprotective effects in rats and mice (Lambert et al., Epilepsia 42:321-27 (2001)). These actions were accompanied by changes in nitric oxide production (Ross et al., Eur. J. Pharmacol. 401:121-30 (2000)) and the expression of pro-inflammatory proteins (Costa et al., Br. J. Pharmacol. 137:413-20 (2002)). N-stearoylethanolamine showed pro-apoptotic and anorexic effects (Okamoto et al., Chem. Biodivers. 4:1842-57 (2007)). It could affect cell signaling and elicit biological effects potentially through targets other than cannabinoid receptors, such as exerting anorexic effects in mice via down-regulation of a liver enzyme expression (Terrazzino et al., FASEB J. 18:1580 (2004)), and having anti-inflammatory activity by a passive IgE-induced cutaneous anaphylaxis (Ezzili et al., Bioorg. Med. Chem. Lett. 20:5959-68 (2010)). N-oleoylethanolamine exerted anorexigenic effects by binding to the nuclear receptor in the periphery tissues, leading to body fat loss (Thabuis et al., Lipids 43:887-94 (2008); Astarita et al., J. Pharmacol. Exp. Ther. 318:563 (2006)).
N-acylethanolamines from fatty acids were typically synthesized by reacting fatty acid chloride (Giuffrida et al., Eur. J. Pharmacol. 408:161-68 (2000); Koutek et al., J. Biol. Chem. 269:22937-40 (1994)), fatty acid methyl ester (Farris, J. Am. Oil Chem. Soc. 56:770-73 (1979); Maag, J. Am. Oil Chem. Soc. 61:259-67 (1984)) or triacylglycerol (Lee et al., J. Am. Oil Chem. Soc. 84:945-52 (2007)) with an alkanolamine, or by a direct reaction between free fatty acid and an alkanolamine in the presence of a catalysts at a low temperature or without any catalyst at a high temperature (Liu et al., J. Agric. Food. Chem. 49:5761-64 (2001)). Yield and purity improvement was usually achieved by removal of water, methanol, glycerol or hydrochloric acid. However, only 60-90% N-acylethanolamines were produced from free fatty acid, fatty acid methyl ester and triacylglycerol at the temperature above 100° C., usually at 180° C. for 6-12 hours (Tufvesson et al., Biotechnol. Bioeng. 97:447-53 (2007)). It has been reported that about 99% N-acylethanolamine was obtained if two moles of fatty acid and one mole of ethanolamine were first reacted at 180° C. to give the N,O-bis-acylethanolamine, which was then transesterified with another mole of ethanolamine to form the N-acylethanolamine (Maag, J. Am. Oil Chem. Soc. 61:259-67 (1984)). Nevertheless, this reaction was conducted at a high temperature, which resulted in the formation of products with dark color that significantly influences the product's quality. The addition of deodorizers and antioxidants has been suggested to improve the quality of final products (Tufvesson et al., Biotechnol. Bioeng. 97:447-53 (2007)). The main problems for synthesis of N-acylethanolamines are the low conversion of the reactants and the esterification reaction occurred during the reaction, which result in a low yield of N-acylethanolamines.
Purity of commercial alkanolamides for surfactant purposes was about 80% (Khanmohammadi et al., J. Surfactants Deterg. 12:37-41 (2009)), but with the increasing knowledge and interest in N-acylethanolamines as lipid mediators in animals, plants, or humans, high purity N-acylethanolamines are needed to validate their biological functions in cellular and animal systems. Currently, this type of study is limited to small animal experiments due to the lack of access to these compounds in a desired purity and quantity. For example, the price of N-stearoyl and N-palmitoylethanolamines from the Sigma Chemical Company is $82/5 mg and $60.8/10 mg. Thus, it is necessary to establish a simple, efficient, effective and economical synthesis method to support the investigation of biological and nutritional properties of N-acylethanolamines in large animals and human subjects.
The present invention is directed to fulfilling these needs in the art.